A phase‐sensitive flow cytometer has been developed that combines flow cytometry and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making phase‐resolved measurements on fluorochrome‐labeled cells and particles. Stained cells are analyzed as they intersect a high‐frequency intensity‐modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using only a single‐channel optical detector. The detector output signals, which are phase shifted from a reference signal and amplitude demodulated, are processed by phase‐sensitive detection electronics to resolve signals from heterogeneous fluorescence emissions and quantify single‐component decay times. Results show signal phase shift and amplitude demodulation on fluorospheres; a detection limit threshold of 300–500 fluorescein molecules equivalence for excitation frequencies 1–30 MHz; a measurement precision (coefficient of variation) of 1.8% on alignment fluorospheres and 3.6% on cells stained for DNA content; the resolution of fluorescence signals from cells stained in combination with two fluorochromes, based on differences in their decay times; and the measurement of fluorescence lifetimes directly by the two‐phase ratio method.